E16.5 C57B6 mouse embryo cortices were dissected and then dissociated in 1× Hank's Balanced Salt Solution (HBSS), 20 mg/ml trypsin (Worthington Biochemicals, Lakewood, NJ), and 0.32 mg/ml L-cysteine (Sigma, St. Louis, MO) for 10 minutes. Trypsin treatment was terminated with three two-minute washes in 1× HBSS with 10 mg/ml trypsin inhibitor (Sigma, St. Louis, MO). Trituration of cells was performed with a flame-narrowed Pasteur pipette to fully dissociate cells. Neurons were seeded at an approximate density of 1×106/well on 6-well culture plates. The dishes were pre-coated overnight with poly-ornithine (30 µg/mL, Sigma) in water, washed three times with water, and washed once with Neurobasal Medium (Life Technologies, Carlsbad, CA) before use. Neurons were maintained in 2 ml/well Neurobasal Medium containing B27 Supplement (2%; Invitrogen, Carlsbad, CA), penicillin-streptomycin (50 µg/ml penicillin, 50 U/ml streptomycin, Sigma) and glutamine (1 mM, Sigma, St. Louis, MO). Neurons were grown in vitro for 7 days. 8 ml of the medium was replaced with 10 ml fresh warm medium on the 4th and 6th days in vitro (DIV).
